Vaccines: Introduction to immunity, Fundamental concepts in vaccination, Production of Modern Vaccines (production of Hepatitis vaccine).

Vaccines

A vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The agent stimulates the body’s immune system to recognize the agent as foreign, destroy it, and “remember” it, so that the immune system can more easily recognize and destroy any of these microorganisms that it later encounters.

Introduction to immunity

The immune system refers to a collection of cells and proteins that function to protect the skin, respiratory passages, intestinal tract and other areas from foreign antigens, such as microbes (organisms such as bacteria, fungi, and parasites), viruses, cancer cells, and toxins. The immune system can be simplistically viewed as having two “lines of defense”: innate immunity and adaptive immunity. Innate immunity represents the first line of defense to an intruding pathogen. It is an antigen-independent (non-specific) defense mechanism that is used by the host immediately or within hours of encountering an antigen. The innate immune response has no immunologic memory and, therefore, it is unable to recognize or “memorize” the same pathogen should the body be exposed to it in the future. Adaptive immunity, on the other hand, is antigen-dependent and antigen-specific and, therefore, involves a lag time between exposure to the antigen and maximal response. The hallmark of adaptive immunity is the capacity for memory which enables the host to mount a more rapid and efficient immune response upon subsequent exposure to the antigen. Innate and adaptive immunity are not mutually exclusive mechanisms of host defense, but rather are complementary, with defects in either system resulting in host vulnerability.

Innate immunity

The primary function of innate immunity is the recruitment of immune cells to sites of infection and inflammation through the production of cytokines (small proteins involved in cell-cell communication). Cytokine production leads to the release of antibodies and other proteins and glycoproteins which activate the complement system, a biochemical cascade that functions to identify and opsonize (coat) foreign antigens, rendering them susceptible to phagocytosis (process by which cells engulf microbes and remove cell debris). The innate immune response also promotes clearance of dead cells or antibody complexes and removes foreign substances present in organs, tissues, blood and lymph. It can also activate the adaptive immune response through a process known as antigen presentation.

Numerous cells are involved in the innate immune response such as phagocytes (macrophages and neutrophils), dendritic cells, mast cells, basophils, eosinophils, natural killer (NK) cells and lymphocytes (T cells). Phagocytes are sub-divided into two main cell types: neutrophils and macrophages. Both of these cells share a similar function: to engulf (phagocytose) microbes. In addition to their phagocytic properties, neutrophils contain granules that, when released, assist in the elimination of pathogenic microbes. Unlike neutrophils (which are short-lived cells), macrophages are long-lived cells that not only play a role in phagocytosis, but are also involved in antigen presentation to T cells. Macrophages are named according to the tissue in which they reside. For example, macrophages present in the liver are called Kupffer cells while those present in the connective tissue are termed histiocytes.

Adaptive immunity

Adaptive immunity develops when innate immunity is ineffective in eliminating infectious agents and the infection is established. The primary functions of the adaptive immune response are the recognition of specific “non-self” antigens in the presence of “self” antigens; the generation of pathogen-specific immunologic effector pathways that eliminate specific pathogens or pathogen-infected cells; and the development of an immunologic memory that can quickly eliminate a specific pathogen should subsequent infections occur. The cells of the adaptive immune system include: T cells, which are activated through the action of antigen presenting cells (APCs), and B cells.

T cells derive from hematopoietic stem cells in bone marrow and, following migration, mature in the thymus. These cells express a unique antigen-binding receptor on their membrane, known as the T-cell receptor (TCR), and as previously mentioned, require the action of APCs (usually dendritic cells, but also macrophages, B cells, fibroblasts and epithelial cells) to recognize a specific antigen.

The surfaces of APCs express cell-surface proteins known as the major histocompatibility complex (MHC). MHC are classified as either class I (also termed human leukocyte antigen [HLA] A, B and C) which are found on all nucleated cells, or class II (also termed HLA, DP, DQ and DR) which are found on only certain cells of the immune system, including macrophages, dendritic cells and B cells. Class I MHC molecules present endogenous (intracellular) peptides while class II molecules present exogenous (extracellular) peptides. The MHC protein displays fragments of antigens (peptides) when a cell is infected with a pathogen or has phagocytosed foreign proteins.

T cells are activated when they encounter an APC that has digested an antigen and is displaying antigen fragments bound to its MHC molecules. The MHC-antigen complex activates the TCR and the T cell secretes cytokines which further control the immune response. This antigen presentation process stimulates T cells to differentiate into either cytotoxic T cells (CD8+ cells) or T-helper (Th) cells (CD4+ cells). Cytotoxic T cells are primarily involved in the destruction of cells infected by foreign agents. They are activated by the interaction of their TCR with peptide-bound MHC class I molecules. Clonal expansion of cytotoxic T cells produce effector cells which release perforin and granzyme (proteins that causes lysis of target cells) and granulysin (a substance that induces apoptosis of target cells). Upon resolution of the infection, most effector cells die and are cleared by phagocytes. However, a few of these cells are retained as memory cells that can quickly differentiate into effector cells upon subsequent encounters with the same antigen.

T helper (Th) cells play an important role in establishing and maximizing the immune response. These cells have no cytotoxic or phagocytic activity, and cannot kill infected cells or clear pathogens. However, they “mediate” the immune response by directing other cells to perform these tasks. Th cells are activated through TCR recognition of antigen bound to class II MHC molecules. Once activated, Th cells release cytokines that influence the activity of many cell types, including the APCs that activate them.

Passive Immunity

Passive immunity is the transfer of antibody produced by one human or other animal to another. Passive immunity provides protection against some infections, but this protec – tion is temporary. The antibodies will degrade during a period of weeks to months, and the recipient will no longer be protected. The most common form of passive immunity is that which an infant receives from its mother. Antibodies are trans – ported across the placenta during the last 1–2 months of pregnancy. As a result, a full-term infant will have the same antibodies as its mother. These antibodies will protect the infant from certain diseases for up to a year.

Protection is better against some diseases (e.g., measles, rubella, tetanus) than others (e.g., polio, pertussis). Many types of blood products contain antibody. Some products (e.g., washed or reconstituted red blood cells) contain a relatively small amount of antibody, and some (e.g., intravenous immune globulin and plasma products) contain a large amount.

In addition to blood products used for transfusion (e.g., whole blood, red cells, and platelets) there are three major sources of antibody used in human medicine. These are homologous pooled human antibody, homologous human hyperimmune globulin, and heterologous hyperimmune serum.

Homologous pooled human antibody is also known as immune globulin. It is produced by combining (pooling) the IgG antibody fraction from thousands of adult donors in the United States. Because it comes from many different donors, it contains antibody to many different antigens. It is used primarily for postexposure prophylaxis for hepatitis A and measles and treatment of certain congenital immuno – globulin deficiencies.

Active Immunity

Active immunity is stimulation of the immune system to produce antigen-specific humoral (antibody) and cellular immunity. Unlike passive immunity, which is temporary, active immunity usually lasts for many years, often for a lifetime.

One way to acquire active immunity is to survive infection with the disease-causing form of the organism. In general, once persons recover from infectious diseases, they will have lifelong immunity to that disease. The persistence of protection for many years after the infection is known as immunologic memory. Following exposure of the immune system to an antigen, certain cells (memory B cells) continue to circulate in the blood (and also reside in the bone marrow) for many years. Upon reexposure to the antigen, these memory cells begin to replicate and produce antibody very rapidly to reestablish protection.

Another way to produce active immunity is by vaccination. Vaccines interact with the immune system and often produce an immune response similar to that produced by the natural infection, but they do not subject the recipient to the disease and its potential complications. Many vaccines also produce immunologic memory similar to that acquired by having the natural disease.

Fundamental concepts in vaccination

Immunology and Vaccine-Preventable Diseases Immunology is a complicated subject, and a detailed discussion of it is beyond the scope of this text. However, an understanding of the basic function of the immune system is useful in order to understand both how vaccines work and the basis of recommendations for their use. The description that follows is simplified. Many excellent immunology textbooks are available to provide additional detail.

Immunity is the ability of the human body to tolerate the presence of material indigenous to the body (“self”), and to eliminate foreign (“nonself”) material. This discriminatory ability provides protection from infectious disease, since most microbes are identified as foreign by the immune system. Immunity to a microbe is usually indicated by the presence of antibody to that organism. Immunity is gener – ally specific to a single organism or group of closely related organisms. There are two basic mechanisms for acquiring immunity, active and passive.

 

 

Traditional methods of vaccine production

The first human vaccines against viruses were based using weaker or attenuated viruses to generate immunity. The smallpox vaccine used cowpox, a poxvirus that was similar enough to smallpox to protect against it but usually didn’t cause serious illness. Rabies was the first virus attenuated in a lab to create a vaccine for humans.

Egg-Based Vaccines

Over the last 60 years, seasonal flu vaccines have been manufactured using fertilized embryonic eggs.  Using this method, it takes about four months to produce a batch of vaccines for a new strain of influenza virus; from the moment the new influenza virus’ culture becomes available for vaccine manufacturing.   The advantages of using embryonic eggs to manufacture seasonal flu vaccines are that the safety and effectiveness of the vaccines produced have been well established.

Cell-Based Vaccines

Since the mid 1990’s, newer vaccine manufacturing methods were developed.  The cell-based vaccine manufacturing process is one of such methods.   The cell-based vaccine manufacturing process uses cells from mammals to culture the influenza virus for vaccine production.  Various pharmaceutical companies use different sources of mammalian cell cultures for the vaccine manufacturing process.  Baxter Healthcare uses cells extracted from the kidney of the African Green Monkey while companies such as Solvay Biologicals and Novartis Vaccines use kidney cells from canines to produce seasonal flu vaccines.

Production of DPT vaccine

DPT is a class of combination vaccines against three infectious diseases in humans: diphtheria, pertussis (whooping cough), and tetanus. The vaccine components include diphtheria and tetanus toxoids and killed whole cells of the bacterium that causes pertussis.

Although different combinations may contain the same toxoids or antigens each vaccine may differ substantially according to the toxoid or antigen dose, number of pertussis components (for acellular vaccines), method of purification and inactivation of the toxins and incorporation of adjuvants and excipients. All of these factors may have an impact on the reactogenicity of different DTP vaccine combinations.

Diphtheria and Tetanus (DT and Td) toxoid combination: DT vaccine used for primary immunisation and boosting in children contains 6.7-25Lf of diphtheria toxoid and 5 – 7.5 Lf of tetanus toxoid per dose. An adult combination, Td, is used for boosting and primary immunisation in adolescents and adults and contains a lower dose of diphtheria (less than 2 Lf/dose) but a similar dose of tetanus toxoid.

Diphtheria, Tetanus and Pertussis (DTP) combinations: Initial DTP combination preparations contained whole-cell pertussis antigens. Concern due to common occurrence of minor local reactions and less common severe reactions of whole-cell pertussis led to the development of acellular vaccines and clinical trials demonstrating their efficacy in the 1980’s. Multiple acellular pertussis vaccines are now available and are referred to by the number of acellular antigen components that they contain. Whole-cell pertussis vaccine remains a safe, inexpensive and effective vaccine which is used in many countries because whole cell vaccines that generate a higher level of antibody to pertussis toxin are associated with higher vaccine efficacy.

DTP with other vaccine antigen combinations: There are many vaccine formulations containing diphtheria and tetanus toxoids and whole cell or acellular pertussis antigens in combination with Haemophilus influenzae type b, hepatitis B and/or inactivated polio virus to produce quadrivalent, pentavalent and hexavalent combination vaccines.

Rabies vaccine

Pre-exposure vaccination should be offered to people at high risk of exposure to rabies, such as laboratory staff working with rabies virus, veterinarians, animal handlers and wildlife officers, and other individuals living in or travelling to countries or areas at risk. Travellers with extensive outdoor exposure in rural areas – such as might occur while running, bicycling, hiking, camping, backpacking, etc. – may be at risk, even if the duration of travel is short. Preexposure vaccination is advisable for children living in or visiting countries or areas at risk, where they provide an easy target for rabid animals. Pre-exposure vaccination is also recommended for individuals travelling to isolated areas or to areas where immediate access to appropriate medical care is limited or to countries where modern rabies vaccines are in short supply and locally available rabies vaccines might be unsafe and/or ineffective.

Pre-exposure rabies vaccination consists of three full intramuscular (i.m.) doses of cell-culture- or embryonated-egg-based vaccine given on days 0, 7 and 21 or 28 (a few days’ variation in the timing is not important). For adults, the vaccine should always be administered in the deltoid area of the arm; for young children (under 1 year of age), the anterolateral area of the thigh is recommended. Rabies vaccine should never be administered in the gluteal area: administration in this manner will result in lower neutralizing antibody titres.

To reduce the cost of cell-derived vaccines for pre-exposure rabies vaccination, intradermal (i.d.) vaccination in 0.1-ml volumes on days 0, 7 and either 21 or 28 may be considered. This method of administration is an acceptable alternative to the standard intramuscular administration, but it is technically more demanding and requires appropriate staff training and qualified medical supervision. Concurrent use of chloroquine can reduce the antibody response to intradermal application of cell-culture rabies vaccines. People who are currently receiving malaria prophylaxis or who are unable to complete the entire three-dose pre-exposure series before starting malarial prophylaxis should therefore receive pre-exposure vaccination by the intramuscular route.

Periodic booster injections are not recommended for general travellers. However, in the event of exposure through the bite or scratch of an animal known or suspected to be rabid, individuals who have previously received a complete series of pre- or post-exposure rabies vaccine (with cell-culture or embryonated-egg vaccine) should receive two booster doses of vaccine. Ideally, the first dose should be administered on the day of exposure and the second 3 days later. This should be combined with thorough wound treatment (see “Post-exposure prophylaxis”, below). Rabies immunoglobulin is not required for patients who have previously received a complete vaccination series.

 

Production of modern vaccines

Live Attenuated Vaccines

Live vaccines are derived from “wild,” or disease-causing, viruses or bacteria. These wild viruses or bacteria are attenuated, or weakened, in a laboratory, usually by repeated culturing. For example, the measles virus used as a vaccine today was isolated from a child with measles disease in 1954. Almost 10 years of serial passage using tissue culture media was required to transform the wild virus into attenuated vaccine virus.

To produce an immune response, live attenuated vaccines must replicate (grow) in the vaccinated person. A relatively  small dose of virus or bacteria is administered, which replicates in the body and creates enough of the organism to stimulate an immune response. Anything that either damages the live organism in the vial (e.g., heat, light) or interferes with replication of the organism in the body (circulating antibody) can cause the vaccine to be ineffective. Although live attenuated vaccines replicate, they usually do not cause disease such as may occur with the “wild” form of the organism. When a live attenuated vaccine does cause “disease,” it is usually much milder than the natural disease and is referred to as an adverse reaction.

The immune response to a live attenuated vaccine is virtu – ally identical to that produced by a natural infection. The immune system does not differentiate between an infection with a weakened vaccine virus and an infection with a wild virus. Live attenuated vaccines produce immunity in most recipients with one dose, except those administered orally. However, a small percentage of recipients do not respond to the first dose of an injected live vaccine (such as MMR or varicella) and a second dose is recommended to provide a very high level of immunity in the population. Live attenuated vaccines may cause severe or fatal reac – tions as a result of uncontrolled replication (growth) of the vaccine virus. This only occurs in persons with immunodefi – ciency (e.g., from leukemia, treatment with certain drugs, or human immunodeficiency virus (HIV) infection).

A live attenuated vaccine virus could theoretically revert to its original pathogenic (disease-causing) form. This is known to happen only with live (oral) polio vaccine. Active immunity from a live attenuated vaccine may not develop because of interference from circulating antibody to the vaccine virus. Antibody from any source (e.g., transpla – cental, transfusion) can interfere with replication of the vaccine organism and lead to poor response or no response to the vaccine (also known as vaccine failure). Measles vaccine virus seems to be most sensitive to circulating antibody. Polio and rotavirus vaccine viruses are least affected. Live attenuated vaccines are fragile and can be damaged or destroyed by heat and light. They must be handled and stored carefully.

Inactivated Vaccines

Inactivated vaccines are produced by growing the bacterium or virus in culture media, then inactivating it with heat and/ or chemicals (usually formalin). In the case of fractional vaccines, the organism is further treated to purify only those components to be included in the vaccine (e.g., the polysac – charide capsule of pneumococcus.) Inactivated vaccines are not alive and cannot replicate. The entire dose of antigen is administered in the injection. These vaccines cannot cause disease from infection, even in an immunodeficient person.

Inactivated antigens are less affected by circulating antibody than are live agents, so they may be given when antibody is present in the blood (e.g., in infancy or following receipt of antibody-containing blood products.) Inactivated vaccines always require multiple doses. In general, the first dose does not produce protective immunity, but “primes” the immune system. A protective immune response develops after the second or third dose. In contrast to live vaccines, in which the immune response closely resembles natural infection, the immune response to an inactivated vaccine is mostly humoral. Little or no cellular immunity results. Antibody titers against inactivated antigens diminish with time. As a result, some inactivated vaccines may require periodic supplemental doses to increase, or “boost,” antibody titers.

Currently available whole-cell inactivated vaccines are limited to inactivated whole viral vaccines (polio, hepatitis A, and rabies). Inactivated whole virus influenza vaccine and whole inactivated bacterial vaccines (pertussis, typhoid, cholera, and plague) are no longer available in the United States. Fractional vaccines include subunits (hepatitis B, influenza, acellular pertussis, human papillomavirus, anthrax) and toxoids (diphtheria, tetanus.) A subunit vaccine for Lyme disease is no longer available in the United States.

Polysaccharide Vaccines

Polysaccharide vaccines are a unique type of inactivated subunit vaccine composed of long chains of sugar molecules that make up the surface capsule of certain bacteria. Pure polysaccharide vaccines are available for three diseases: pneumococcal disease, meningococcal disease, and Salmonella Typhi.

The immune response to a pure polysaccharide vaccine is typically T-cell independent, which means that these vaccines are able to stimulate B cells without the assistance of T-helper cells. T-cell–independent antigens, including polysaccharide vaccines, are not consistently immunogenic in children younger than 2 years of age. Young children do not respond consistently to polysaccharide antigens, probably because of immaturity of the immune system.

Repeated doses of most inactivated protein vaccines cause the antibody titer to go progressively higher, or “boost.” This does not occur with polysaccharide antigens; repeat doses of polysaccharide vaccines usually do not cause a booster response. Antibody induced with polysaccharide vaccines has less functional activity than that induced by protein antigens. This is because the predominant antibody produced in response to most polysaccharide vaccines is IgM, and little IgG is produced.

Recombinant Vaccines

Vaccine antigens may also be produced by genetic engi – neering technology. These products are sometimes referred to as recombinant vaccines. Four genetically engineered vaccines are currently available in the United States. Hepatitis B and human papillomavirus (HPV) vaccines are produced by insertion of a segment of the respective viral gene into the gene of a yeast cell or virus. The modified yeast cell produces pure hepatitis B surface antigen or HPV capsid protein when it grows. Live typhoid vaccine (Ty21a) is Salmonella Typhi bacteria that have been genetically modified to not cause illness. Live attenuated influenza vaccine has been engineered to replicate effectively in the mucosa of the nasopharynx but not in the lungs.

Production of Hepatitis Vaccine

Hepatitis B vaccine is a vaccine that prevents hepatitis B. The first dose is recommended within 24 hours of birth with either two or three more doses given after that. This includes those with poor immune function such as from HIV/AIDS and those born premature. It is also recommended for health-care workers to be vaccinated. In healthy people routine immunization results in more than 95% of people being protected.

Blood testing to verify that the vaccine has worked is recommended in those at high risk. Additional doses may be needed in people with poor immune function but are not necessary for most people. In those who have been exposed to the hepatitis B virus but not immunized, hepatitis B immune globulin should be given in addition to the vaccine.The vaccine is given by injection into a muscle.

n 1963, the American physician/geneticist Baruch Blumberg discovered what he called the “Australia Antigen” (now called HBsAg) in the serum of an Australian Aboriginal person. In 1968, this protein was found to be part of the virus that causes “serum hepatitis” (hepatitis B) by virologist Alfred Prince. The American microbiologist/vaccinologist Maurice Hilleman at Merck used three treatments (pepsin, urea and formaldehyde) of blood serum together with rigorous filtration to yield a product that could be used as a safe vaccine. Hilleman hypothesized that he could make an HBV vaccine by injecting patients with hepatitis B surface protein. In theory, this would be very safe, as these excess surface proteins lacked infectious viral DNA. The immune system, recognizing the surface proteins as foreign, would manufacture specially shaped antibodies, custom-made to bind to, and destroy, these proteins. Then, in the future, if the patient were infected with HBV, the immune system could promptly deploy protective antibodies, destroying the viruses before they could do any harm.

The first large-scale trials for the blood-derived vaccine were performed on gay men, in accordance with their high-risk status. Later, Hilleman’s vaccine was falsely blamed for igniting the AIDS epidemic. But, although the purified blood vaccine seemed questionable, it was determined to have indeed been free of HIV. The purification process had destroyed all viruses—including HIV. The vaccine was approved in 1981.

The blood-derived hepatitis B vaccine was withdrawn from the marketplace in 1986 when Pablo DT Valenzuela, Research Director of Chiron Corporation, succeeded in making the antigen in yeast and invented the world’s first recombinant vaccine. The recombinant vaccine was developed by inserting the HBV gene that codes for the surface protein into the yeast Saccharomyces cerevisiae. This allows the yeast to produce only the noninfectious surface protein, without any danger of introducing actual viral DNA into the final product. This is the vaccine still in use today.

 

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